Altered april binding antibodies

ABSTRACT

The invention relates to APRIL-binding antibodies, which bind the same epitope of human APRIL as an antibody having an antigen binding site of hAPRIL.01A. The antibodies of the present invention comprise specific selections of framework sequences of the V H  and V L  domains and have unexpected features in comparison to hAPRIL.01A. The invention further relates to compositions comprising an antibody of the invention and to the medical and diagnostic uses of the antibodies and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/978,699, filed May 14, 2018, now U.S. Pat. No. 10,377,830, which is adivisional of U.S. patent application Ser. No. 14/991,708, filed Jan. 8,2016, now U.S. Pat. No. 9,969,808, issued May 15, 2018, which claims thebenefit of Dutch Patent Application No. NL 2014108, filed Jan. 9, 2015,and issued as Dutch Patent No. NL2014108 on Sep. 30, 2016, each of whichis hereby incorporated in its entirety including all tables, figures,and claims.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 12, 2019, isnamed ABE-0001-CT_SeqListing.txt and is 73 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to isolated antibodies, includingfragments thereof, which bind human APRIL, to polynucleotides encodingsuch antibodies and host cells producing said antibodies. The antibodiescan be used to treat cancers and inhibit immune cell proliferation andconditions that may benefit from such inhibition of immune cellproliferation such as autoimmune diseases, inflammatory diseases, ordiseases associated with immunoglobulin over production. In addition,the antibodies can be used as diagnostic tool and in vitro agents forinhibition of immune cell proliferation and/or survival.

BACKGROUND OF THE INVENTION

APRIL is expressed as a type-II transmembrane protein, but unlike mostother TNF family members it is mainly processed as a secreted proteinand cleaved in the Golgi apparatus where it is cleaved by a furinconvertase to release a soluble active form (Lopez-Fraga et al., 2001,EMBO Rep 2:945-51). APRIL assembles as a non-covalently linkedhomo-trimer with similar structural homology in protein fold to a numberof other TNF family ligands (Wallweber et al., 2004, Mol Biol 343,283-90). APRIL binds two TNF receptors: B cell maturation antigen (BCMA)and transmembrane activator and calcium modulator and cyclophilin ligandinteractor (TACI) (reviewed in Kimberley et al., 2009, J Cell Physiol.218(1):1-8). In addition, APRIL has recently been shown to bind heparansulphate proteoglycans (HSPGs) (Hendriks et al., 2005, Cell Death Differ12, 637-48). APRIL has been shown to have a role in B cell signallingand drive both proliferation and survival of human and murine B cellsin-vitro (reviewed in Kimberley et al., 2009, J Cell Physiol.218(1):1-8).

APRIL is predominantly expressed by immune cell subsets such asmonocytes, macrophages, dendritic cells, neutrophils, B-cells, andT-cells, many of which also express BAFF. In addition, APRIL can beexpressed by non-immune cells such as osteoclasts, epithelial cells anda variety of tumour tissues (reviewed in Kimberley et al., 2009, J CellPhysiol. 218(1):1-8). In fact, APRIL was originally identified based onits expression in cancer cells (Hahne et al., 1998, J Exp Med 188,1185-90). High expression levels of APRIL mRNA were found in a panel oftumour cell lines as well as human primary tumours such as colon, and alymphoid carcinoma.

A retrospective study under 95 Chronic Lymphocytic Leukaemia (CLL) CLLpatients showed increased levels of APRIL in serum, which correlatedwith disease progression and overall patient survival, with a poorerprognosis for patients with high APRIL serum levels (Planelles et al.,2007, Haematologica 92, 1284-5). Similarly, (increased levels of) APRILwas shown to be expressed in Hodgkin's lymphoma, Non-Hodgkin's lymphoma(NHL) and Multiple Myeloma (MM) (reviewed in Kimberley et al., 2009, JCell Physiol. 218(1):1-8). A retrospective study in DLBCL patients (NHL)showed that high APRIL expression in cancer lesions correlated with apoor survival rate (Schwaller et al., 2007, Blood 109, 331-8). Recently,APRIL serum levels in serum from patients suffering from colorectalcancer were shown to have a positive diagnostic value (Ding et al.,2013, Clin. Biochemistry, dx.doi.org/10.1016/j.clinbiochem.2013.06.008).

Due to its role in B cell biology APRIL also plays a role in manyautoimmune diseases. Increased serum levels of APRIL have been reportedin many SLE patients (Koyama et al., 2005, Ann Rheum Dis 64, 1065-7). Aretrospective analysis revealed that APRIL serum levels tended tocorrelate with anti-dsDNA antibody titres. Also in the synovial fluid ofpatients with inflammatory arthritis significantly increased APRILlevels as compared with those with patients suffering fromnon-inflammatory arthritis such as osteoarthritis were detected (Stohlet al., 2006, Endocr Metab Immune Disord Drug Targets 6, 351-8; Tan etal., 2003, Arthritis Rheum 48, 982-92).

Several studies focused on the presence of APRIL in the sera of patientssuffering from a wider range of systemic immune-based rheumatic diseases(now also including Sjögren's syndrome, Reiter's syndrome, psoriaticarthritis, polymyositis, and ankylosing spondylitis) and foundsignificantly increased APRIL levels in these patients, suggesting animportant role for APRIL in these diseases as well (Jonsson et al.,1986, Scand J Rheumatol Suppl 61, 166-9; Roschke et al., 2002, J Immunol169, 4314-21). In addition, increased APRIL serum levels were detectedin serum from patients suffering atopic dermatitis (Matsushita et al.,2007, Exp. Dermatology 17, 197-202). Also, serum APRIL levels areelevated in sepsis and predict mortality in critically ill patients(Roderburg et al., J. Critical Care, 2013,dx.doi.org/10.1016/j.jcrc.2012.11.007). Furthermore, APRIL serum levelswere found to be increased in patients suffering from IgA nephropathy(McCarthy et al., 2011, J. Clin. Invest. 121(10):3991-4002).

Finally, increased APRIL expression has also been linked to MultipleSclerosis (MS). APRIL expression was found to be increased in theastrocytes of MS sufferers compared with normal controls. This is inline with the described APRIL expression in glioblastomas and in theserum of glioblastoma patients (Deshayes et al., 2004, Oncogene 23,3005-12; Roth et al., 2001, Cell Death Differ 8, 403-10).

APRIL plays a crucial role in the survival and proliferative capacity ofseveral B-cell malignancies, and potentially also some solid tumours.APRIL is also emerging as a key player in inflammatory diseases orautoimmunity. Thus, strategies to antagonise APRIL are a therapeuticgoal for a number of these diseases. Indeed clinical studies targetingAPRIL with TACI-Fc (Atacicept) are currently ongoing for treatment ofseveral autoimmune diseases. However, TACI-Fc also targets BAFF, afactor involved in normal B-cell maintenance. Antibodies directedagainst APRIL have been described in WO9614328, WO2001/60397,WO2002/94192, WO9912965, WO2001/196528, WO9900518 and WO2010/100056.WO2010/100056 describes antibodies targeting APRIL specifically. Theantibodies of WO2010/100056 fully block the binding of APRIL to TACI andat least partially to BCMA. Antibody hAPRIL.01A fully blocks the bindingto both BCMA and TACI. The hAPRIL.01A antibody inhibited B-cellproliferation, survival and antigen-specific Immunoglobulin secretion invitro and in vivo (Guadagnoli et al., 2011, Blood 117(25):6856-65). Inaddition, hAPRIL.01A inhibited proliferation and survival of malignantcells in in vitro and in vivo representative of human CLL and MM disease(Guadagnoli et al., 2011, Blood 117(25):6856-65; Lascano et al., 2013,Blood 122(24): 3960-3; Tai et al., 2014, ASH poster 2098). Finally,hAPRIL.01A inhibited the secretion of antigen-specific IgA (Guadagnoliet al., 2011, Blood 117(25):6856-65). In view of these unique bindingfeatures this murine antibody has a unique pharmaceutical utility.However, in view of its murine origin there are also certain drawbacksin the pharmaceutical utility of this antibody in human medicine. Thepresent invention therefore is aimed at providing altered hAPRIL.01Aantibodies more suitable for use in human medicine.

SUMMARY OF THE INVENTION

The present invention provides hAPRIL.01A analogues comprising certainsubstitutions of the framework regions of the V_(H) and V_(L) domains.It has been surprisingly found that when in an antigen binding site ofhAPRIL.01A the framework regions of the V_(H) domain of hAPRIL.01A aresubstituted for framework regions from a V_(H) amino acid sequenceselected from SEQ ID NO 12, 14, 16 or 18 and the framework regions ofthe V_(L) domain of hAPRIL.01A are substituted for the framework regionsof a V_(L) amino acid sequence selected from SEQ ID NO 30, functionalhAPRIL.01A analogues are obtained. This is surprising in view of thefact that research by the inventors of the present invention has shown,that only limited combinations of alternative V_(H) and V_(L) frameworksequences from human origin can support adequate binding of thehAPRIL.01A CDRs to human APRIL and thus can result in functionalhAPRIL.01A analogues. In addition the inventors of the present inventionhave shown that further improvements in the hAPRIL.01 analogues may beobtained by introducing certain specific amino acid substitutions. Inparticular amino acid substitution R72S and/or the double substitutionR67K in combination with V68A in a selected V_(H) amino acid sequence.

The invention thus according to a first aspect relates to anAPRIL-binding antibody, binding to the same epitope of human APRIL as anantibody, having an antigen binding site of hAPRIL.01A, such asmonoclonal antibody hAPRIL.01A disclosed in WO2010/100056, said humanAPRIL-binding antibody comprising a number of antigen binding sitescomprising V_(H) and V_(L) domains, wherein in an antigen binding sitethe framework sequences of the V_(H) domain have at least 70% sequencesimilarity with the framework sequences of a V_(H) amino acid sequenceselected from SEQ ID NO: 12, 14, 16 or 18, preferably with SEQ ID NO: 14or 18, most preferably SEQ ID NO: 18, and the framework sequences of theV_(L) domain have at least 70% sequence similarity with the frame worksequences of a V_(L) amino acid sequence selected from SEQ ID NO: 30.

Further aspect of the invention relate to polynucleotides, in isolatedform, coding for the variable region of the heavy chain and light chainof the antibody of the invention, an expression unit comprising a numberof the polynucleotides and a host cell comprising the expression unitand/or a number of the polynucleotides.

Yet a further aspect of the invention relates to a method of producingan antibody of the invention, which method comprises:

-   -   a) culturing a host cell of the invention in culture medium        under conditions wherein the number of polynucleotides is        expressed, thereby producing polypeptides comprising the light        and heavy chain variable regions; and    -   b) recovering the polypeptides from the host cell or culture        medium.

A composition comprising an antibody of the invention in combinationwith a pharmaceutically acceptable carrier or diluent and optionally anumber of other active compounds is the subject of a further aspect ofthe invention.

The therapeutic and diagnostic use of the antibody of the invention isyet another aspect of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES

The sequences presented in the sequence listing relate to the amino acidsequences and encoding DNA sequences of V_(H) and V_(L) domains and ofheavy and light chains from which framework sequences may be employed inthe antibodies according to the invention. In addition the amino acidsequences of the CDRs of both the V_(H) and V_(L) domains of hAPRIL.01Aand of the heavy and light chains are presented. According to certainembodiments of the invention CDRs of hAPRIL.01A are employed in theantibody of the invention. Table 1 below correlates the sequence IDs totheir respective sequence.

TABLE 1 Sequence Listing SEQ ID NO: Description 1 hAPRIL.01A heavy chainvariable region (DNA) 2 hAPRIL.01A light chain variable region (DNA) 3hAPRIL.01A heavy chain variable region (AA) 4 hAPRIL.01A light chainvariable region (AA) 5 hAPRIL.01A heavy chain CDR1 (AA) 6 hAPRIL.01Aheavy chain CDR2 (AA) 7 hAPRIL.01A heavy chain CDR3 (AA) 8 hAPRIL.01Alight chain CDR1 (AA) 9 hAPRIL.01A light chain CDR2 (AA) 10 hAPRIL.01Alight chain CDR3 (AA) 11 VH11 heavy chain variable region (DNA) 12 VH11heavy chain variable region (AA) 13 VH12 heavy chain variable region(DNA) 14 VH12 heavy chain variable region (AA) 15 VH13 heavy chainvariable region (DNA) 16 VH13 heavy chain variable region (AA) 17 VH14heavy chain variable region (DNA) 18 VH14 heavy chain variable region(AA) 19 VL10 light chain variable region (DNA) 20 VL10 light chainvariable region (AA) 21 VL11 light chain variable region (DNA) 22 VL11light chain variable region (AA) 23 VL12 light chain variable region(DNA) 24 VL12 light chain variable region (AA) 25 VL13 light chainvariable region (DNA) 26 VL13 light chain variable region (AA) 27 VL14light chain variable region (DNA) 28 VL14 light chain variable region(AA) 29 VL15 light chain variable region (DNA) 30 VL15 light chainvariable region (AA) 31 VH14_1 heavy chain variable region (DNA) 32VH14_1 heavy chain variable region (AA) 33 VH14_1C heavy chain variableregion (DNA) 34 VH14_1C heavy chain variable region (AA) 35 VH14_1Dheavy chain variable region (DNA) 36 VH14_1D heavy chain variable region(AA) 37 VH14_1E heavy chain variable region (DNA) 38 VH14_1E heavy chainvariable region (AA) 39 VH14_1G heavy chain variable region (DNA) 40VH14_1G heavy chain variable region (AA) 41 VH11 heavy chain (DNA) 42VH11 heavy chain (AA) 43 VH12 heavy chain (DNA) 44 VH12 heavy chain (AA)45 VH13 heavy chain (DNA) 46 VH13 heavy chain (AA) 47 VH14 heavy chain(DNA) 48 VH14 heavy chain (AA) 49 VL15 light chain (DNA) 50 VL15 lightchain (AA) 51 VH14_1G heavy chain (DNA) 52 VH14_1G heavy chain (AA) 53hAPRIL.01A heavy chain (DNA) 54 hAPRIL.01A light chain (DNA) 55hAPRIL.01A heavy chain (AA) 56 hAPRIL.01A light chain (AA) 57 heavychain secretion leader sequence (DNA) 58 heavy chain secretion leadersequence (AA) 59 Light chain secretion leader sequence (DNA) 60 Lightchain secretion leader sequence (AA)

SEQ ID NO: 11-52 relate to engineered immunoglobulin VH, VL, heavy orlight chain sequences as indicated.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D: FIG. 1A shows the results of targeting APRIL withhAPRIL.01A or the analogue 14_1G.15 in an in vivo test for T-cellindependent B cell response. Transgenic mice (APRIL-Tg) were challengedwith 250 μg NP-Ficoll (day 0), and treated with hAPRIL.01A or 14_1G.15on day −1 and 3. PBS and wildtype mice (WT) were used as negativecontrols. IgA1 immunoglobulin titres were measured by ELISA. 14_1G.15inhibited the T-cell independent immune response to NP-Ficoll moreefficacious then its hAPRIL.01A analogue. FIG. 1B shows the results oftargeting APRIL with hAPRIL.01A or the analogue 14_1G.15 in an in vivotest for T-cell independent B cell response. Transgenic mice (APRIL-Tg)were challenged with 250 μg NP-Ficoll (day 0), and treated withhAPRIL.01A or 14_1G.15 on day −1 and 3. PBS and wildtype mice (WT) wereused as negative controls. IgA2 immunoglobulin titres were measured byELISA. 14_1G.15 inhibited the T-cell independent immune response toNP-Ficoll more efficacious then its hAPRIL.01A analogue. FIG. 1C showsthe results of targeting APRIL with hAPRIL.01A or the analogue 14_1G.15in an in vivo test for T-cell independent B cell response. Transgenicmice (APRIL-Tg) were challenged with 250 μg NP-Ficoll (day 0), andtreated with hAPRIL.01A or 14_1G.15 on day −1 and 3. PBS and wildtypemice (WT) were used as negative controls. IgG immunoglobulin titres weremeasured by ELISA. 14_1G.15 inhibited the T-cell independent immuneresponse to NP-Ficoll more efficacious then its hAPRIL.01A analogue.FIG. 1D shows the results of targeting APRIL with hAPRIL.01A or theanalogue 14_1G.15 in an in vivo test for T-cell independent B cellresponse. Transgenic mice (APRIL-Tg) were challenged with 250 μgNP-Ficoll (day 0), and treated with hAPRIL.01A or 14_1G.15 on day −1 and3. PBS and wildtype mice (WT) were used as negative controls. IgMimmunoglobulin titres were measured by ELISA. 14_1G.15 inhibited theT-cell independent immune response to NP-Ficoll more efficacious thenits hAPRIL.01A analogue.

DETAILED DESCRIPTION

The invention thus relates to antibodies that bind to the same epitopeof human APRIL as an antibody, including an antibody analog, such as anantibody fragment, having an antigen binding site of hAPRIL.01A. Theantibody hAPRIL.01A has been disclosed in WO2010/100056 with sequencesof V_(H) and V_(L) domains and CDRs. The inventors of the presentinvention have found that there are limited possibilities to substitutethe mouse framework region of the V_(H) and V_(L) domains of hAPRIL.01Aby human alternatives. In addition the selected alternative frameworksequences results in alternative anti-human APRIL antibodies havingunexpected features. It is believed that the selected frameworksequences manifest their special features within the context of bindingto the human APRIL epitope for hAPRIL.01A and thus have a broad utilitywithin this context. Therefore, the present invention is aimed at anyantibody (including fragments and/or derivatives and/or analogues) thatbinds to the same epitope as hAPRIL.01A and which comprises the selectedframework sequences in its V_(H) and V_(L) domains. In certainembodiments such an antibody will comprise alternative CDRs differentfrom the CDRs of hAPRIL.01A. However, according to other embodiments theantibody will comprise CDRs similar to or even identical to those ofhAPRIL.01A. According to certain embodiments an antibody comprising theV_(H) domain CDR1, CDR2 and CDR3 and the V_(L) domain CDR1, CDR2 andCDR3 of hAPRIL.01A or variants of any of said sequences is to beconsidered an antibody, binding to the same epitope of human APRIL asmonoclonal antibody hAPRIL.01A. This in particular when it binds humanAPRIL with a K_(D) of about 100 nM or lower and/or blocks binding ofhuman APRIL to human BCMA and TACI with an IC₅₀ of about 100 nM orlower. Within the present invention preferred antibodies bind humanAPRIL with a K_(D) value of about 100 nM or lower, such as within aninterval selected from 100-0.001 nM, for example 100-0.010, 100-0.050,100-0.100, 100-0.150, 100-0.200, 100-0.250, 100-0.300, 100-0.350,100-0.400, 100-0.450, 90-0.500, 80-0.550, 70-0.600, 60-0.650, 50-0.700,40-0.750, 30-0.800, 20-0.850, 10-0.900 or 1.0-0.950 nM. As the skilledperson will understand lower values are preferred for the K_(D), such asvalues below 50, 20, 10, 1.00 nM. Antibodies having K_(D) values withinsuch intervals are suitable for clinical applications (See, e.g. Presta,et al., 2001, Thromb. Haemost. 85:379-389; Yang, et al., 2001, Crit.Rev. Oncol. Hematol. 38:17-23; Carnahan, et al., 2003, Clin. Cancer Res.(Suppl.) 9:3982s-3990s). Antibody affinities may be determined usingstandard analysis known to the skilled person, for example asexemplified in the experimental section.

It is further preferred if an antibody of the invention blocks bindingof human APRIL to human BCMA and TACI with an IC₅₀ value of about 100 nMor lower, such as within an interval selected from 100-0.001 nM, forexample 100-0.010, 100-0.050, 100-0.100, 100-0.150, 100-0.200,100-0.250, 100-0.300, 100-0.350, 100-0.400, 100-0.450, 90-0.500,80-0.550, 70-0.600, 60-0.650, 50-0.700, 40-0.750, 30-0.800, 20-0.850,10-0.900 or 1.0-0.950 nM. As the skilled person will understand lowervalues are preferred for the IC₅₀, such as values below 50, 20, 10, 1.00nM.

Binding of the antibody to the same epitope as hAPRIL.01A may beevaluated by assessing the binding competition for human APRIL of anantibody of the present invention and a reference antibody having anantigen binding site of hAPRIL.01A in accordance with the methodspresented in example 2 or 5 of WO2010/100056 or other cross-blocking orepitope mapping techniques known to the skilled person as discussedbelow. The antigen binding site of hAPRIL.01A is defined by the V_(H)and V_(L) domains as presented in SEQ ID NO: 3 and 4. Thus any antibody,including an antibody analog, such as an antibody fragment comprisingthe V_(H) and V_(L) domains as presented in SEQ ID NO: 3 and 4 may beused as a reference antibody for evaluating the binding to the sameepitope of human APRIL as hAPRIL.01A. Antibody hAPRIL.01A, disclosed inWO2010/100056 is an example of an antibody having an antigen bindingsite of hAPRIL.01A and is a suitable reference antibody within thecontext of the present invention. However, also antibody fragments, suchas Fab, F(ab)₂ or Fv fragments derived from hAPRIL.01A may be used as areference antibody. Based on the DNA sequences of SEQ ID NO: 1 and 2 andthe amino acid sequences of SEQ ID NO: 3 and 4, the skilled person willbe able to construct and produce such antibody fragments derived fromhAPRIL.01A. Based on the provided sequences the skilled person will alsobe able to produce hAPRIL.01A analogs for use as reference antibodies byjoining the heavy chain variable region amino acid sequence of SEQ IDNO: 3 (coded by SEQ ID NO: 1) to the IgG1 constant region (from themouse or from a different species, preferably from the mouse) andjoining the light chain variable region amino acid sequence of SEQ IDNO: 4 (coded by SEQ ID NO: 2) to the κ constant region (from the mouseor from a different species, preferably from the mouse).

When evaluated with such methods, antibodies binding to the same epitopeof human APRIL as hAPRIL.01A may block binding of a reference antibodyhaving an hAPRIL.01A antigen binding site to human APRIL with an IC₅₀ ofabout 100 nM or lower, such as within an interval selected from100-0.001 nM, for example 100-0.010, 100-0.050, 100-0.100, 100-0.150,100-0.200, 100-0.250, 100-0.300, 100-0.350, 100-0.400, 100-0.450,90-0.500, 80-0.550, 70-0.600, 60-0.650, 50-0.700, 40-0.750, 30-0.800,20-0.850, 10-0.900 or 1.0-0.950 nM. As the skilled person willunderstand lower values are preferred for the IC₅₀, such as values below50, 20, 10, 1.00 nM.

The antibody of the invention thus may have one or more of the followingfeatures:

-   -   (i) binds human APRIL with a K_(D) of about 100 nM or lower;    -   (ii) blocks binding of human APRIL to human BCMA and human TACI        with an IC₅₀ of about 100 nM or lower;    -   (iii) blocks binding of hAPRIL.01A to human APRIL with an IC₅₀        of about 100 nM or lower.

These features may be combined in the following combinations: (i) or(ii) or (iii); (i) and (ii); (i) and (iii); (ii) and (iii); (i) and (ii)and (iii).

According to the present invention the framework sequences of the V_(H)domain of an antigen binding site of the antibody are selected such thatthey have at least 70% sequence similarity with the framework sequencesof a V_(H) amino acid sequence selected from SEQ ID NO. 12, 14, 16 or18. According to a preferred embodiment the framework sequences of aV_(H) domain of the antibody are selected such that they have at least70% sequence similarity with the framework sequences of a V_(H) aminoacid sequence selected from SEQ ID NO. 14 or 18, most preferably SEQ IDNO: 18. The framework sequences of the V_(L) domain in said antigenbinding site of the antibody are selected such that they have at least70% sequence similarity with the framework sequences of a V_(L) aminoacid sequence selected from SEQ ID NO. 30.

The V_(H) amino acid sequence selected from SEQ ID NO. 12, 14, 16 or 18and the V_(L) amino acid sequence selected from SEQ ID NO. 30, compriseboth framework sequences and CDR sequences. The CDR sequencesincorporated in these V_(H) and V_(L) amino acid sequences are those ofhAPRIL.01A, and correspond to SEQ ID NO. 5 (hAPRIL.01A V_(H) CDR1), 6(hAPRIL.01A V_(H) CDR2), 7 (hAPRIL.01A V_(H) CDR3), 8 (hAPRIL.01A V_(L)CDR1), 9 (hAPRIL.01A V_(L) CDR2), 10 (hAPRIL.01A V_(L) CDR3). However,as already stated above, the use of the V_(H) and V_(L) frameworksequences, as selected within the present invention, is not restrictedto combination with the specific CDRs of hAPRIL.01A. Thus the sequencesimilarity of at least 70% according to certain embodiments is to beconsidered for the framework sequences only and not for the full V_(H)amino acid sequence as selected from SEQ ID NO. 12, 14, 16, 18, or thefull V_(L) amino acid sequence as selected from SEQ ID NO. 30. Theframework sequences for the V_(H) amino acid sequences as presented inSEQ ID NO. 12, 14, 16 or 18 are the parts of these sequences outside theV_(H) CDRs i.e. the parts outside the sequence parts identical to SEQ IDNO. 5 (hAPRIL.01A V_(H) CDR1), 6 (hAPRIL.01A V_(H) CDR2), 7 (hAPRIL.01AV_(H) CDR3). The framework sequences for the V_(L) amino acid sequenceas presented in SEQ ID NO. 30 are the parts of this sequence outside theV_(L) CDRs i.e. the parts outside the sequence parts identical to SEQ IDNO. 8 (hAPRIL.01A V_(L) CDR1), 9 (hAPRIL.01A V_(L) CDR2), 10 (hAPRIL.01AV_(L) CDR3).

According to alternative embodiments the sequence similarity of at least70% is to be considered for the full V_(H) amino acid sequence asselected from SEQ ID NO. 12, 14, 16 or 18 and the full V_(L) amino acidsequence as selected from SEQ ID NO. 30.

Within the description of the present invention at least 70% sequencesimilarity should be understood as meaning at least 80%, such as atleast 85%, preferably at least 90%, more preferably at least 95%, suchas at least 99% sequence similarity.

As the skilled person will understand, “sequence similarity” refers tothe extent to which individual nucleotide or peptide sequences arealike. The extent of similarity between two sequences is based on theextent of identity combined with the extent of conservative changes. Thepercentage of “sequence similarity” is the percentage of amino acids ornucleotides which is either identical or conservatively changed viz.“sequence similarity”=(% sequence identity)+(% conservative changes).

For the purpose of this invention “conservative changes” and “identity”are considered to be species of the broader term “similarity”. Thus,whenever the term sequence “similarity” is used it embraces sequence“identity” and “conservative changes”. According to certain embodimentsthe conservative changes are disregarded and the % sequence similarityrefers to % sequence identity.

The term “sequence identity” is known to the skilled person. In order todetermine the degree of sequence identity shared by two amino acidsequences or by two nucleic acid sequences, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in thesequence of a first amino acid or nucleic acid sequence for optimalalignment with a second amino or nucleic acid sequence). Such alignmentmay be carried out over the full lengths of the sequences beingcompared. Alternatively, the alignment may be carried out over a shortercomparison length, for example over about 20, about 50, about 100 ormore nucleic acids/bases or amino acids.

The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide positions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The degree of identityshared between sequences is typically expressed in terms of percentageidentity between the two sequences and is a function of the number ofidentical positions shared by identical residues in the sequences (i.e.,% identity=number of identical residues at corresponding positions/totalnumber of positions×100). Preferably, the two sequences being comparedare of the same or substantially the same length.

The percentage of “conservative changes” may be determined similar tothe percentage of sequence identity. However, in this case changes at aspecific location of an amino acid or nucleotide sequence that arelikely to preserve the functional properties of the original residue arescored as if no change occurred.

For amino acid sequences the relevant functional properties are thephysico-chemical properties of the amino acids. A conservativesubstitution for an amino acid in a polypeptide of the invention may beselected from other members of the class to which the amino acidbelongs. For example, it is well-known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity and hydrophilicity) can be substituted for another aminoacid without substantially altering the activity of a protein,particularly in regions of the protein that are not directly associatedwith biological activity (see, e.g., Watson, et al., Molecular Biologyof the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987)).For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andtyrosine. Polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Conservative substitutions include, for example, Lys forArg and vice versa to maintain a positive charge; Glu for Asp and viceversa to maintain a negative charge; Ser for Thr and vice versa so thata free —OH is maintained; and Gln for Asn and vice versa to maintain afree —NH₂.

Exemplary conservative substitutions in the amino acid sequence of theCD70 binding peptides of the invention can be made in accordance withthose set forth below as follows:

TABLE 2 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys, His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

For nucleotide sequences the relevant functional properties is mainlythe biological information that a certain nucleotide carries within theopen reading frame of the sequence in relation to the transcriptionand/or translation machinery. It is common knowledge that the geneticcode has degeneracy (or redundancy) and that multiple codons may carrythe same information in respect of the amino acid for which they code.For example in certain species the amino acid leucine is coded by UUA,UUG, CUU, CUC, CUA, CUG codons (or TTA, TTG, CTT, CTC, CTA, CTG forDNA), and the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU,AGC (or TCA, TCG, TCC, TCT, AGT, AGC for DNA). Nucleotide changes thatdo not alter the translated information are considered conservativechanges.

The skilled person will be aware of the fact that several differentcomputer programs, using different mathematical algorithms, areavailable to determine the identity between two sequences. For instance,use can be made of a computer program employing the Needleman and Wunschalgorithm (Needleman et al. (1970)). According to an embodiment thecomputer program is the GAP program in the Accelrys GCG software package(Accelrys Inc., San Diego U.S. A). Substitution matrices that may beused are for example a BLOSUM 62 matrix or a PAM250 matrix, with a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6. The skilled person will appreciate that all these differentparameters will yield slightly different results but that the overallpercentage identity of two sequences is not significantly altered whenusing different algorithms.

According to an embodiment the percent identity between two nucleotidesequences is determined using the GAP program in the Accelrys GCGsoftware package (Accelrys Inc., San Diego U.S. A) A NWSgapdna CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6 is used.

In another embodiment, the percent identity of two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (Meyers et al. (1989)) which has been incorporated into theALIGN program (version 2.0) (available at the ALIGN Query using sequencedata of the Genestream server IGH Montpellier Francexylian.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weight residuetable, a gap length penalty of 12 and a gap penalty of 4.

For the present invention it is most preferred to use BLAST (Basic LocalAlignment Tool) to determine the percentage identity and/or similaritybetween nucleotide or amino acid sequences.

Queries using the BLASTn, BLASTp, BLASTx, tBLASTn and tBLASTx programsof Altschul et al. (1990) may be posted via the online versions of BLASTaccessible via http://www.ncbi.nlm.nih.gov. Alternatively a standaloneversion of BLAST {e.g., version 2.2.29 (released 3 Jan. 2014))downloadable also via the NCBI internet site may be used. PreferablyBLAST queries are performed with the following parameters. To determinethe percentage identity and/or similarity between amino acid sequences:algorithm: blastp; word size: 3; scoring matrix: BLOSUM62; gap costs:Existence: 11, Extension: 1; compositional adjustments: conditionalcompositional score matrix adjustment; filter: off; mask: off. Todetermine the percentage identity and/or similarity between nucleotidesequences: algorithm: blastn; word size: 11; max matches in query range:0; match/mismatch scores: 2, −3; gap costs: Existence: 5, Extension: 2;filter: low complexity regions; mask: mask for lookup table only.

The percentage of “conservative changes” may be determined similar tothe percentage of sequence identity with the aid of the indicatedalgorithms and computer programs. Some computer programs, e.g., BLASTp,present the number/percentage of positives (=similarity) and thenumber/percentage of identity. The percentage of conservative changesmay be derived therefrom by subtracting the percentage of identity fromthe percentage of positives/similarity (percentage conservativechanges=percentage similarity−percentage identity).

As the skilled person will understand, the antibody of the inventionwill comprise a number of antigen binding sites. Framework sequences ofthe amino acid sequences of the selected V_(H) and V_(L) domainstogether with CDR sequences are combined in an antigen binding site.Specific combinations of framework sequences from V_(H) and V_(L)domains envisaged by the present invention are as presented in Table 3below, wherein an “X” is presented at a position of a combination of aV_(H) and V_(L) domain envisaged.

TABLE 3 V_(H) and V_(L) combination of framework sequences. V_(H) SEQ IDNO 12 14 16 18 32 34 36 38 40 V_(L) SEQ ID NO. 30 X X X X X X X X X

The combination of framework sequences from these V_(H) and V_(L)domains results in antibodies having functional binding to human APRIL.It should be noted, as is further presented in the experimental section,that in the tests performed by the inventors of the present invention,antibodies having APRIL binding functionality were only obtained, whencombining the VH sequences of the invention with the VL sequence of SEQID NO: 30 (VL15). In the tested combinations of the selected VHsequences with other VL sequences (VL10-VL14), the obtained antibodiesdid not have functional APRIL binding properties. A further surprisingeffect of the combination of the VH and VL sequences used in accordancewith the present invention is an improved (thermo)stability incomparison to antibodies having the hAPRIL.01A VH and VL sequences as ispresented in the experimental section.

There is a preference for combining the VL framework sequences of SEQ IDNO: 30 with the VH framework sequences from the VH framework sequencesof SEQ ID NO: 18 or sequences derived therefrom, such as SEQ ID NO 32,34, 36, 38, 40. These preferred combinations are presented with anunderlined “X” in Table 3. The most preferred combination of VLframework sequences from SEQ ID NO: 30 with VH framework sequences fromSEQ ID NO: 40 is presented in table 3 as an “X” in bold (and underlined)font. It has been surprisingly found that these combinations of VL andVH framework sequences result in antibodies having additional beneficialfeatures, including beneficial stability features and/or improvedbinding to the human APRIL target.

According to certain embodiments in the V_(H) domain at least one ofCDR1, CDR2, CDR3 is selected from the group consisting of respectivelySEQ NO 5, 6, 7, or a variant of any of said sequences. Preferably in theV_(H) domain, CDR1, CDR2 and CDR3 are selected from respectively SEQ NO5, 6, 7, or a variant of any of said sequences. These V_(H) domain CDRsequences correspond to the V_(H) domain CDRs of hAPRIL.01A.

According to certain embodiments in the V_(L) domain at least one ofCDR1, CDR2, CDR3 is selected from the group consisting of respectivelySEQ NO 8, 9, 10, or a variant of any of said sequences. Preferably inthe V_(L) domain CDR1, CDR2 and CDR3 are selected from respectively SEQNO 8, 9, 10, or a variant of any of said sequences. These V_(L) domainCDR sequences correspond to the V_(L) domain CDRs of hAPRIL.01A.

According to certain embodiments in an antigen binding site of theantibody the V_(H) domain CDR1, CDR2 and CDR3 are selected fromrespectively SEQ NO 5, 6, 7, or a variant of any of said sequences andthe V_(L) domain CDR1, CDR2 and CDR3 of are selected from respectivelySEQ NO 8, 9, 10, or a variant of any of said sequences.

The inventors of the present invention have surprisingly found thatfurther improvements can be made in the antibodies combining the VH andVL framework sequences used in the present invention. In particular thesubstitution R72S in the VH amino acid sequence results in improvedbinding to human APRIL.

In addition the combined substitution R67K-V68A in the VH amino acidsequence also results in improved binding to human APRIL. The inventiontherefore according to certain embodiments relates to antibodies whereinin the V_(H) amino acid sequence the amino acid at position 72 is S. TheVH amino sequences of SEQ ID NO. 32, 34, 36, 38, 40 are examples of suchVH amino acid sequences having an S residue at position 72. According toother embodiments the invention relates to antibodies wherein in theV_(H) amino acid sequence the amino acid at position 67 is K and theamino acid at position 68 is A. The combination of all three amino acidsubstitutions R72S, R67K and V68A is also envisaged within the presentinvention. Thus according to other embodiments the invention relates toantibodies wherein in the V_(H) amino acid sequence the amino acid atposition 72 is S, the amino acid at position 67 is K and the amino acidat position 68 is A.

Apart from V_(H) and V_(L) domains, the antibody may comprise additionaldomains such as a suitable number of C_(H) domains and a suitable numberof C_(L) domains. C_(H) domains and C_(L) domains may be of humanorigin. Such domains also include domains that provide antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g. U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta, 2006, Adv. Drug Delivery Rev. 58:640-656;Vincent and Zurini, Biotechnol. J., 2012, 7:1444-50; Kaneko and Niwa,Biodrugs, 2011, 25: 1-11. Such modification can be used to enhance orsuppress various reactions of the immune system, with possiblebeneficial effects in diagnosis and therapy. Alterations of the Fcregion include amino acid changes (substitutions, deletions andinsertions), glycosylation or deglycosylation, and adding multiple Fc.According to certain embodiments it is preferred to use Fc regionsdisplaying reduced Fc effector functions. The antibodies of the presentinvention according to certain embodiments may also have Fc regionsoriginating from human IgG4 and/or Fc regions carrying a N297Qglycosylation deficient mutant. C_(L) domains may be selected from humanKappa or Lamba constant domains. Preferably, human Kappa C_(L) domain isused.

According to certain embodiments of the invention, antibodies comprisingFc and C_(L) domains are provided, wherein the V_(H) domain amino acidsequence is in a heavy chain amino acid sequence having at least 70%sequence similarity with an amino acid sequence selected from SEQ ID NO:42, 44, 46, 48, 52 preferably SEQ ID NO: 48 or 52, most preferably SEQID NO: 52, and the V_(L) domain amino acid sequence is in a light chainamino acid sequence having at least 70% sequence similarity with anamino acid sequence selected from SEQ ID NO: 50.

Specific combinations of these heavy and light chains envisaged by thepresent invention are as presented in Table 4 below, wherein an “X” ispresented at a position of a combination of a heavy and light chainenvisaged.

TABLE 4 Heavy chain SEQ ID NO 42 44 46 48 52 Light chain SEQ 50 X X X XX ID NO.

Preferred combinations are indicated with an underlined “X” in. Morepreferred combinations are indicated with an “X” in bold (andunderlined) font.

According to a further aspect, the invention relates to an isolatedpolynucleotide encoding a V_(H) domain and/or a V_(L) domain of anantibody according to the invention. A polynucleotide sequence encodingthe V_(H) domain preferably is a polynucleotide sequence having at least70% sequence similarity with a polynucleotide sequence selected from SEQID NO: 11, 13, 15, 17, 31, 39, 41, 43, 45, 47, 51 preferably SEQ ID NO:17, 31, 39, 47 or 51, more preferably SEQ ID NO: 51. A polynucleotidesequence encoding the V_(L) domain preferably is a polynucleotidesequence having at least 70% sequence similarity with a polynucleotidesequence selected from SEQ ID NO: 29 or 49, preferably SEQ ID NO: 49.

The invention further relates to an expression unit comprising a numberof expression vectors, comprising a number of polynucleotides accordingto the invention under the control of suitable regulatory sequences,wherein the number of polynucleotides encode the V_(H) domain and theV_(L) domain of an antibody according to the invention. The expressionunit may be designed such that the polynucleotide sequence coding forthe V_(H) domain and the polynucleotide sequence coding for V_(L) domainmay be on the same expression vector. Thus the expression unit maycomprise a single vector. Alternatively the polynucleotide sequencecoding for the V_(H) domain and the polynucleotide sequence coding forthe V_(L) domain may be on different expression vectors. In suchembodiments the expression unit will comprise a plurality, such as forexample 2, expression vectors.

A further aspect of the invention relates to a host cell comprising anumber of polynucleotides of the invention and/or an expression unit ofthe invention. The expression unit preferably is an expression unitcomprising an expression vector comprising both a polynucleotidesequence coding for the V_(H) domain and a polynucleotide sequencecoding for the V_(L) domain.

-   -   The antibody of the invention can be any one of the following:        -   a chimeric antibody or a fragment thereof;        -   a humanized antibody or a fragment thereof; or        -   an antibody fragment selected from the group consisting of            Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′)₂, bispecific mAb and a            diabody. Humanized antibodies comprising a number of antigen            binding sites based on the CDRs of hAPRIL.01A are preferred.            It may be noted that the framework regions selected            according to the invention are from human origin and thus            may be suitably used for obtaining humanized antibodies, in            particular when combined with constant regions from human            origin.

According to a further aspect thereof, the invention relates to a methodof producing an antibody of the invention, which method comprises:

-   -   a) culturing a host cell comprising a number of polynucleotides        of the invention and/or an expression unit of the invention in        culture medium under conditions wherein the polynucleotide is        expressed, thereby producing polypeptides comprising the light        and heavy chain variable regions; and    -   b) recovering the polypeptides from the host cell or culture        medium.

The invention further relates to a composition comprising an antibody ofthe invention in combination with a pharmaceutically acceptable carrieror diluent. Such composition in one embodiment may comprise more thanone antibody. In one embodiment, the composition comprises one or moreother active compounds in addition to the one or more antibodies of theinvention. Such combination compositions can be used for combinationtherapy, for example in the treatment of cancer. In that case theantibody may be combined with one or more of the usual anticancer drugs.For other combination therapies other additional active compounds may beused. For combination therapy it is not obligatory to have the two ormore active compounds in the same composition. Thus, also part of theinvention is the combined or subsequent use of the antibodies and theother active compound, wherein the antibody and the other activecompound are administered simultaneously or subsequently.

As is clear from the description above, the antibody of the inventionmay be for use in therapy and diagnosis and for other, non-therapeuticpurposes. The invention thus further relates to methods of use of theantibodies in therapy and diagnosis and for other, non-therapeuticpurposes.

In one embodiment, the therapy comprises inhibition of immune cellproliferation and/or immune cell survival. In another embodiment thetreatment is aimed at treating cancer. In one embodiment, the therapycomprises the treatment of an autoimmune disease. In one embodiment, thetherapy comprises the treatment of an inflammatory disease. In oneembodiment, the therapy comprises the treatment of an Ig secretionmediated disease, in particular an IgA secretion mediated disease. Thetherapeutic uses of the antibody of the invention will be discussed inmore detail below.

The antibody of the invention when used in non-therapeutic applicationscan for example be applied in in vitro or ex vivo techniques, such asflow-cytometry, Western blotting, enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.

Therapy

In view of the fact that the antibodies of the present invention bind tohuman APRIL analogous to hAPRIL.01A, the antibodies of the presentinvention are suitable for use in therapy analogous to hAPRIL.01A, withthe improvements discussed above and in the experimental section.Therefore, the antibodies of the present invention are suitable fortreatment of a condition known or expected to be ameliorated by blockingthe interaction of human APRIL with BCMA and/or TACI. As is alreadyknown in the art, blocking the interaction of human APRIL with BCMAand/or TACI inhibits immune cell proliferation and/or survival and thusmay be of value for the treatment of conditions where such blocking ofimmune cell proliferation and/or survival is beneficial, such asinflammatory diseases, diseases mediated by Ig secretion and/orautoimmune diseases. Blocking of the interaction of human APRIL withBCMA and/or TACI may also be beneficial in the treatment of cancer.

Autoimmune conditions for which an antibody of the invention may bebeneficial may be selected from multiple sclerosis, rheumatoidarthritis, type 1 diabetes, psoriasis, Crohn's disease and otherinflammatory bowel diseases such as ulcerative colitis, systemic lupuseythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis(MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, Gravesdisease, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, scleroderma with anti-collagen antibodies, mixed connectivetissue disease, polypyositis, pernicious anemia, idiopathic Addison'sdisease, autoimmune associated infertility, glomerulonephritis,crescentic glomerulonephritis, proliferative glomerulonephritis, bullouspemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin resistance,autoimmune diabetes mellitus, autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmunehepatitis, autoimmune hemophilia, autoimmune lymphoproliferativesyndrome, autoimmune uveoretinitis, Guillain-Bare syndrome,arteriosclerosis and Alzheimer's disease.

In addition, the antibodies of the invention may also be beneficial inthe treatment of other conditions associated where lowering of immuneresponses is beneficial, such as graft (transplant) rejection orallergic conditions.

Also, the antibodies of the invention may be beneficial in the treatmentof other conditions wherein lowering of Immunoglobulin levels, such asIgA, including IgA1 or IgA2, IgG, IgM levels, is beneficial, such asconditions associated with Ig secretion, in particular IgA secretion, Igoverproduction, such as IgA, including IgA1 or IgA2, IgG, IgM overproduction, in particular IgA overproduction, or Ig deposition, inparticular IgA deposition. Examples of such conditions include, but arenot limited to IgA nephropathy and other forms of glomerulonephritis,celiac disease, pemphigoid diseases, Henloch-Schönlein purpura, andother autoimmune diseases that are associated with Ig deposition.

Within the present invention the treatment of the “condition” includesany therapeutic use including prophylactic and curative uses of theanti-human APRIL antibody. Therefore the term “condition” may refer todisease states but also to physiological states in the prophylacticsetting where physiology is not altered to a detrimental state.

Cancers within the present invention include, but are not limited to,leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,myeloblasts promyelocyte, myelomonocytic monocytic erythroleukemia,chronic leukemia, chronic myelocytic (granulocytic) leukemia, chroniclymphocytic leukemia, mantle cell lymphoma, primary central nervoussystem lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma,Polycythemia vera Lymphoma, Hodgkin's disease, non-Hodgkin's disease,multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma,liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteosarcoma,chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem. Cancers that are of particular interest are cancers having cellsexpressing APRIL, such as B-cell derived malignancies, lymphoid or colonor lung carcinoma's or multiple myeloma (MM) or Chronic LymphocyticLeukaemia (CLL) B cells.

For the purpose of treatment of any of the conditions mentioned above,the antibody of the invention can be dosed directly to subjects, aloneor in combination with other therapeutic agents. Therefore, according tocertain embodiments of the invention an antibody of the invention in itsuse and/or in a composition may be combined with a number ofchemotherapeutic agents, which are used to treat multiple myeloma (MM),Myelodysplastic syndrome (MDS), Waldenströms macroglobinemia, B-CLL,Diffuse large cell B cell lymphoma, Non-Hodgkin Lymphoma and wegenersgranulomatosis, such as melphalan, vincristine, fludarabine,chlorambucil, bendamustine, etoposide, doxorubicin, cyclophosphamide,cisplatin. In addition, an antibody of the invention in its use and/orin a composition may be combined with a number of immune modulatingagents such as corticosteroids (dexamethasone, prednisolone),thalidomide analogs (thalidomide, lenalidomide, pomalidomide). Also anantibody of the invention in its use and/or in a composition may becombined with a number of targeted kinase inhibitors, such as ibrutinib,idelalisib. Furthermore, an antibody of the invention in its use and/orin a composition may be combined with a number of antibody therapiestargeting CD20, such as rituximab, ofatumumab, obinotuzumab; or antibodytherapies targeting CD52 such as alemtuzumab; or antibody therapiestargeting CD38 such as daratumumab; or antibody therapies targeting IL-6or IL-6 receptor (such as sarilumab, tocilizumab); or antibody therapiestargeting CS-1 (such as elotuzumab); or antibody therapies targetingBCMA (such as GSK2857916); or antibody therapies targeting BAFF or BLyss(such as tabalumab). In addition, an antibody of the invention in itsuse and/or in a composition may be combined with a numberbisphosphonates (such as pamidronate, zolendronic acid). It is describedpreviously that APRIL protects MM cells from IL-6 deprivation,dexamethasone and bortezomib treatment (Moreaux et al, 2004, Blood103(8): 3148-57; Li et al., 2010, Med Oncol. 27:439-45). hAPRIL.01A hasbeen shown to reverse the APRIL mediated survival of MM cells inlenalidomide and dexamethasone treatment (Tai et al., 2014, ASH poster2098). In view of these findings in the art, the antibody of the presentinvention may in particular be combined in its use and/or in acomposition with a further therapeutic agent selected fromcorticosteroids, for example dexamethasone, prednisolone, preferablydexamethasone, or thalidomide analogs, for example thalidomide,lenalidomide, pomalidomide, in particular lenalidomide, or withbortezomid.

Diagnosis

With APRIL representing an important marker for diseases, such as, butnot limited to autoimmune diseases, inflammatory diseases andmalignancies, detection of APRIL in the serum and/or tissue of humansubjects is important. For diagnostic applications, the antibodiestypically will be labeled (either directly or indirectly) with adetectable moiety. Numerous labels are available which can be generallygrouped into the following categories: biotin, fluorochromes,radionucleotides, enzymes, iodine, and biosynthetic labels.

Soluble APRIL present in the serum and other body fluids and/or tissueof a range of different patients has been shown to correlate withdisease severity of the patients. For example, patients suffering fromchronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, Non-Hodgkin'slymphoma (NHL) and Multiple Myeloma (MM), DLBCL patients (NHL),colorectal cancer, SLE, a wider range of systemic immune-based rheumaticdiseases (now also including Sjögren's syndrome, Reiter's syndrome,psoriatic arthritis, polymyositis, and ankylosing spondylitis) andatopic dermatitis demonstrated increased serum levels of soluble APRIL.In addition, serum APRIL levels in patients suffering from IgAnephropathy are elevated (McCarthy et al., 2011, J. Clin. Invest.121(10):3991-4002). Also, serum APRIL levels are elevated in sepsis andpredict mortality in critically ill patients (Jonsson et al., 1986,Scand J Rheumatol Suppl 61, 166-9; Roschke et al., 2002, J Immunol 169,4314-21). Based on the demonstrated binding characteristics ofhAPRIL.01A, antibodies according to the invention can be used as adiagnostic tool to detect soluble APRIL in the body fluids and/ortissue.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies. A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibodies of the invention may also be used for in vivo diagnosticassays. Generally, the antibody is labeled with a radionuclide so thatthe antigen or cells expressing it can be localized usingimmunoscintigraphy or positron emission tomography.

Non-Therapeutic Uses

According to another aspect of the invention, the antibodies have other,non-therapeutic uses. The non-therapeutic uses for the antibodies of theinvention include flow cytometry, western blotting, enzyme linkedimmunosorbant assay (ELISA) and immunohistochemistry.

The antibodies of this invention may for example be used as an affinitypurification reagent via immobilization to a Protein A-Sepharose column.

General Definitions

The term “antibody” refers to any form of antibody that exhibits thedesired biological activity, such as inhibiting binding of a ligand toits receptor, or by inhibiting ligand-induced signaling of a receptor.In the present case the biological activity comprises blocking of thebinding of APRIL to its receptors BCMA and/or TACI. Thus, “antibody” isused in the broadest sense and specifically covers, but is not limitedto, monoclonal antibodies (including full length monoclonal antibodies)and multispecific antibodies (e.g., bispecific antibodies) such as basedon the Duobody® technology (Genmab) or Hexabody® technology (Genmab) orantibody fragment.

“Antibody fragment” and “antibody binding fragment” mean antigen-bindingfragments and analogues of an antibody, typically including at least aportion of the antigen binding or variable regions (e.g. one or moreCDRs) of the parental antibody. An antibody fragment retains at leastsome of the binding specificity of the parental antibody. Typically, anantibody fragment retains at least 10% of the parental binding activitywhen that activity is expressed on a molar basis. Preferably, anantibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100%or more of the parental antibody's binding affinity for the target.Examples of antibody fragments include, but are not limited to, Fab,Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules, e.g., sc-Fv, unibodies (technology fromGenmab); nanobodies (technology from Ablynx); domain antibodies(technology from Domantis); and multispecific antibodies formed fromantibody fragments. Engineered antibody variants are reviewed inHolliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

An “Fab fragment” is comprised of one light chain and the CH1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

An “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the V_(H) domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form a F(ab′)₂ molecule.

An “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

A “single-chain Fv antibody” (or “scFv antibody”) refers to antibodyfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V_(H)and V_(L) domains which enables the scFv to form the desired structurefor antigen binding. For a review of scFv, see Pluckthun, 1994, ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315. See also, InternationalPatent Application Publication No. WO 88/01649 and U.S. Pat. Nos.4,946,778 and 5,260,203.

A “diabody” is a small antibody fragment with two antigen-binding sites.The fragments comprises a heavy chain variable domain (V_(H)) connectedto a light chain variable domain (V_(L)) in the same polypeptide chain(V_(H)-V_(L) or V_(L)-V_(H)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,e.g., EP 404,097; WO 93/11161; and Holliger et al., 1993, Proc. Natl.Acad. Sci. USA 90: 6444-6448.

“Duobodies” are bispecific antibodies with normal IgG structures(Labrijn et al., 2013, Proc. Natl. Acad. Sci. USA 110 (13): 5145-5150).

“Hexabodies” are antibodies that while retaining regular structure andspecificity have an increased killing ability (Diebolder et al., 2014,Science 343(6176):1260-3).

A “domain antibody fragment” is an immunologically functionalimmunoglobulin fragment containing only the variable region of a heavychain or the variable region of a light chain. In some instances, two ormore V_(H) regions are covalently joined with a peptide linker to createa bivalent domain antibody fragment. The two V_(H) regions of a bivalentdomain antibody fragment may target the same or different antigens.

As used herein antibody hAPRIL.01A is a mouse antibody wherein the heavychain has the amino acid sequence of SEQ ID NO: 55 and the light chainhas the amino acid sequence of SEQ ID NO: 56.

An antibody fragment of the invention may comprise a sufficient portionof the constant region to permit dimerization (or multimerization) ofheavy chains that have reduced disulfide linkage capability, for examplewhere at least one of the hinge cysteines normally involved ininter-heavy chain disulfide linkage is altered as described herein. Inanother embodiment, an antibody fragment, for example one that comprisesthe Fc region, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC (antibody dependentcellular cytotoxicity) function, and/or complement binding (for example,where the antibody has a glycosylation profile necessary for ADCCfunction or complement binding).

The term “chimeric” antibody refers to antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(See, for example, U.S. Pat. No. 4,816,567 and Morrison et al., 1984,Proc. Natl. Acad. Sci. USA 81:6851-6855).

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies contain minimalsequence derived from non-human immunoglobulin. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody optionallyalso will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. The humanizedforms of rodent antibodies will essentially comprise the same CDRsequences of the parental rodent antibodies, although certain amino acidsubstitutions may be included to increase affinity, increase stabilityof the humanized antibody, or for other reasons.

The antibodies of the present invention also include antibodies withmodified (or blocked) Fc regions to provide altered effector functions.See, e.g. U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571;WO2006/0057702; Presta, 2006, Adv. Drug Delivery Rev. 58:640-656. Suchmodification can be used to enhance or suppress various reactions of theimmune system, with possible beneficial effects in diagnosis andtherapy. Alterations of the Fc region include amino acid changes(substitutions, deletions and insertions), glycosylation ordeglycosylation, and adding multiple Fc. Changes to the Fc can alsoalter the half-life of antibodies in therapeutic antibodies, and alonger half-life would result in less frequent dosing, with theconcomitant increased convenience and decreased use of material. SeePresta, 2005, J. Allergy Clin. Immunol. 116:731 at 734-35.

The antibodies of the present invention also include antibodies withintact Fc regions that provide full effector functions, e.g. antibodiesof isotype IgG1, which induce complement-dependent cytotoxicity (CDC) orantibody dependent cellular cytotoxicity (ADCC) in the a targeted cell.

The antibodies may also be conjugated (e.g., covalently linked) tomolecules that improve stability of the antibody during storage orincrease the half-life of the antibody in vivo. Examples of moleculesthat increase the half-life are albumin (e.g., human serum albumin) andpolyethylene glycol (PEG). Albumin-linked and PEGylated derivatives ofantibodies can be prepared using techniques well known in the art. See,e.g. Chapman, 2002, Adv. Drug Deliv. Rev. 54:531-545; Anderson andTomasi, 1988, J. Immunol. Methods 109:37-42; Suzuki et al., 1984,Biochim. Biophys. Acta 788:248-255; and Brekke and Sandlie, 2003, NatureRev. 2:52-62.

The term “hypervariable region,” as used herein, refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR,” defined by sequencealignment, for example residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (see Kabat et al., 1991,Sequences of proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md.) and/or thoseresidues from a “hypervariable loop” (HVL), as defined structurally, forexample, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain (see Chothia and Leskl, 1987, J. Mol. Biol.196:901-917).

“Framework” or “FR” residues or sequences are those variable domainresidues or sequences other than the CDR residues as herein defined.

The antibody of the invention according to certain embodiments may be anisolated antibody. An “isolated” antibody is one that has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials that would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, theantibody will be purified (1) to greater than 95% by weight of antibodyas determined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “monoclonal antibody” when used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., 1975,Nature 256:495, or may be made by recombinant DNA methods (see, forexample, U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may alsobe isolated from phage antibody libraries using the techniques describedin Clackson et al., 1991, Nature 352:624-628 and Marks et al., 1991, J.Mol. Biol. 222:581-597, for example. The monoclonal antibodies hereinspecifically include “chimeric” antibodies.

As used herein, the term “immune cell” includes cells that are ofhematopoietic origin and that play a role in the immune response. Immunecells include lymphocytes, such as B cells and T cells, natural killercells, myeloid cells, such as monocytes, macrophages, eosinophils, mastcells, basophils, and granulocytes.

As used herein, an “immunoconjugate” refers to an anti-human APRILantibody, or a fragment thereof, conjugated to a therapeutic moiety,such as a bacterial toxin, a cytotoxic drug or a radiotoxin. Toxicmoieties can be conjugated to antibodies of the invention using methodsavailable in the art.

As used herein, a sequence “variant” or “variant sequence” refers to asequence that differs from the disclosed sequence at one or more aminoacid residues but which retains the biological activity of the parentmolecule. The invention includes the variants of antibodies explicitlydisclosed by the various sequences. For the V_(H) domain CDR1, CDR2 andCDR3 sequences, according to some embodiments, variant sequences maycomprise up to 6 amino acid substitutions, such as 1, 2, 3, 4, 5 or 6amino acid substitutions, for the CDR1, CDR2 and CDR3 sequences takentogether. Similarly for the V_(L) domain CDR1, CDR2 and CDR3 sequences,according to some embodiments, variant sequences may comprise up to 6amino acid substitutions, such as 1, 2, 3, 4, 5 or 6 amino acidsubstitutions, for the CDR1, CDR2 and CDR3 sequences taken together.

“Conservatively modified variants” or “conservative amino acidsubstitution” refers to substitutions of amino acids are known to thoseof skill in this art and may be made generally without altering thebiological activity of the resulting molecule. Those of skill in thisart recognize that, in general, single amino acid substitutions innon-essential regions of a polypeptide do not substantially alterbiological activity (see, e.g., Watson, et al., Molecular Biology of theGene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition 1987)). Suchexemplary substitutions are preferably made in accordance with those setforth above in Table 2.

As used herein, the term “about” refers to a value that is within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e. the limitations of the measurement system.For example, “about” can mean within 1 or more than 1 standard deviationper the practice in the art. Alternatively, “about” or “comprisingessentially of” can mean a range of up to 20%. Furthermore, particularlywith respect to biological systems or processes, the terms can mean upto an order of magnitude or up to 5-fold of a value. When particularvalues are provided in the application and claims, unless otherwisestated, the meaning of “about” or “comprising essentially of” should beassumed to be within an acceptable error range for that particularvalue.

The term “a number of” should be understood as meaning one or more.Depending on the context of its use “a number of” may refer to anysuitable number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Accordingto certain embodiments “a number of” may have the meaning of “aplurality”. Depending on the context of its use “a plurality” may referto any suitable number selected from 2, 3, 4, 5, 6, 7 8, 9, 10.

“Specifically” binds, when referring to a ligand/receptor,antibody/antigen, or other binding pair, indicates a binding reactionwhich is determinative of the presence of the protein, e.g., APRIL, in aheterogeneous population of proteins and/or other biologics. Thus, underdesignated conditions, a specified ligand/antigen binds to a particularreceptor/antibody and does not bind in a significant amount to otherproteins present in the sample.

“Administration”, “therapy” and “treatment,” as it applies to an animal,human, experimental subject, cell, tissue, organ, or biological fluid,refers to contact of an exogenous pharmaceutical, therapeutic,diagnostic agent, or composition to the animal, human, subject, cell,tissue, organ, or biological fluid. “Administration”, “therapy” and“treatment” can refer, e.g., to therapeutic, pharmacokinetic,diagnostic, research, and experimental methods. Treatment of a cellencompasses contact of a reagent to the cell, as well as contact of areagent to a fluid, where the fluid is in contact with the cell.“Administration”, “therapy” and “treatment” also mean in vitro and exvivo treatments, e.g., of a cell, by a reagent, diagnostic, bindingcomposition, or by another cell. Within the present description of theinvention the terms “in vitro” and “ex vivo” have a similar meaning andmay be used interchangeably.

The antibody DNA also may be modified, for example, by substituting thecoding sequence for human heavy- and light-chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, et al., 1984, Proc. Natl Acad. Sci. USA, 81:6851), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for non-immunoglobulin material (e.g., proteindomains). Typically such non-immunoglobulin material is substituted forthe constant domains of an antibody, or is substituted for the variabledomains of one antigen-combining site of an antibody to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Amino acid sequence variants of the anti-human APRIL antibodies of theinvention are prepared by introducing appropriate nucleotide changesinto the coding DNAs, or by peptide synthesis. Such variants include,for example, deletions from, and/or insertions into, and/orsubstitutions of, residues within the amino acid sequences shown for theanti-APRIL antibodies. Any combination of deletion, insertion, andsubstitution is made to arrive at the final construct, provided that thefinal construct possesses the desired characteristics. The amino acidchanges also may alter post-translational processes of the anti-APRILantibodies, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theanti-APRIL antibodies polypeptides that are preferred locations formutagenesis is called “alanine scanning mutagenesis,” as described byCunningham and Wells, 1989, Science 244: 1081-1085. Here, a residue orgroup of target residues are identified (e.g., charged residues such asArg, Asp, His, Lys, and Glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with APRIL antigen. The amino acidresidues demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, Ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-APRILantibodies' variants are screened for the desired activity.

Ordinarily, amino acid sequence variants of the anti-APRIL antibodieswill have an amino acid sequence having at least 75% amino acid sequencesimilarity with the original antibody amino acid sequences of either theheavy or the light chain more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, and most preferably at least95%, 98% or 99%. Similarity or homology with respect to this sequence isas defined above.

Antibodies having the characteristics identified herein as beingdesirable can be screened for increased biologic activity in vitro orsuitable binding affinity. To screen for antibodies that bind to thesame epitope on human APRIL as hAPRIL.01A, a routine cross-blockingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988), can beperformed. Antibodies that bind to the same epitope are likely tocross-block in such assays, but not all cross-blocking antibodies willnecessarily bind at precisely the same epitope since cross-blocking mayresult from steric hindrance of antibody binding by antibodies bind atoverlapping epitopes, or even nearby non-overlapping epitopes.

Alternatively, epitope mapping, e.g., as described in Champe et al.,1995, J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest. “Alanine scanningmutagenesis,” as described by Cunningham and Wells, 1989, Science 244:1081-1085, or some other form of point mutagenesis of amino acidresidues in human APRIL may also be used to determine the functionalepitope for anti-APRIL antibodies of the present invention.

Another method to map the epitope of an antibody is to study binding ofthe antibody to synthetic linear and CLIPS peptides that can be screenedusing credit-card format mini PEPSCAN cards as described by Slootstra etal. (Slootstra et al., 1996, Mol. Diversity 1: 87-96) and Timmerman etal. (Timmerman et al., 2007, J. Mol. Recognit. 20: 283-299). The bindingof antibodies to each peptide is determined in a PEPSCAN-basedenzyme-linked immuno assay (ELISA).

Additional antibodies binding to the same epitope as hAPRIL.01A may beobtained, for example, by screening of antibodies raised against APRILfor binding to the epitope, or by immunization of an animal with apeptide comprising a fragment of human APRIL comprising the epitopesequences. Antibodies that bind to the same functional epitope might beexpected to exhibit similar biological activities, such as similar APRILbinding and BCMA and TACI blocking activity, and such activities can beconfirmed by functional assays of the antibodies.

The antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is anIgG antibody. Any isotype of IgG can be used, including IgG1, IgG2,IgG3, and IgG4. Variants of the IgG isotypes are also contemplated. Theantibody may comprise sequences from more than one class or isotype.Optimization of the necessary constant domain sequences to generate thedesired biologic activity is readily achieved by screening theantibodies in the biological assays described in the Examples.

Likewise, either class of light chain can be used in the compositionsand methods herein. Specifically, kappa, lambda, or variants thereof areuseful in the present compositions and methods.

The antibodies and antibody fragments of the invention may also beconjugated with cytotoxic payloads such as cytotoxic agents orradionucleotides such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I,¹¹C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu,⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn,⁵²Tr and ⁵⁶Fe. Such antibody conjugates may be used in immunotherapy toselectively target and kill cells expressing a target (the antigen forthat antibody) on their surface. Exemplary cytotoxic agents includericin, vinca alkaloid, methotrexate, Psuedomonas exotoxin, saporin,diphtheria toxin, cisplatin, doxorubicin, abrin toxin, gelonin andpokeweed antiviral protein.

The antibodies and antibody fragments of the invention may also beconjugated with fluorescent or chemilluminescent labels, includingfluorophores such as rare earth chelates, fluorescein and itsderivatives, rhodamine and its derivatives, isothiocyanate,phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde,fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label,isoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridimium salt label, an oxalate ester label, an aequorinlabel, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels andstable free radicals.

Any method known in the art for conjugating the antibody molecules orprotein molecules of the invention to the various moieties may beemployed, including those methods described by Hunter et al., 1962,Nature 144:945; David et al., 1974, Biochemistry 13:1014; Pain et al.,1981, J. Immunol. Meth. 40:219; and Nygren, J., 1982, Histochem. andCytochem. 30:407. Methods for conjugating antibodies and proteins areconventional and well known in the art.

Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal., 1992, Bio/Technology 10:163-167 describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc region that is present in the antibody. Protein A canbe used to purify antibodies that are based on human Ig.gamma1,Ig.gamma2, or Ig.gamma4 heavy chains (Lindmark et al., 1983, J. Immunol.Meth. 62:1-13). Protein G is recommended for all mouse isotypes and forhuman .gamma.3 (Guss et al., 1986, EMBO J 5:1567-1575). The matrix towhich the affinity ligand is attached is most often agarose, but othermatrices are available.

Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In one embodiment, the glycoprotein may be purified using adsorptiononto a lectin substrate (e.g. a lectin affinity column) to removefucose-containing glycoprotein from the preparation and thereby enrichfor fucose-free glycoprotein.

Pharmaceutical Formulations

The invention comprises pharmaceutical formulations of an anti-humanAPRIL antibody. To prepare pharmaceutical or sterile compositions, theantibody, in particular an antibody or fragment thereof, is admixed witha pharmaceutically acceptable carrier or excipient, see, e.g.,Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: NationalFormulary, Mack Publishing Company, Easton, Pa. (1984). Formulations oftherapeutic and diagnostic agents may be prepared by mixing withphysiologically acceptable carriers, excipients, or stabilizers in theform of, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions (see, e.g., Hardman, et al., 2001, Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro, 2000, Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.),1993, Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990,Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with another agent, such as theusual anti-cancer drugs, can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio between LD₅₀ and ED₅₀. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

Suitable routes of administration include parenteral administration,such as intramuscular, intravenous, or subcutaneous administration andoral administration. Administration of antibodies, used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral, intraarterial or intravenousinjection. In one embodiment, the antibody of the invention isadministered intravenously. In another embodiment, the antibody of theinvention is administered subcutaneously.

Alternatively, one may administer the antibody in a local rather thansystemic manner, for example, via injection of the antibody directlyinto the site of action, often in a depot or sustained releaseformulation. Furthermore, one may administer the antibody in a targeteddrug delivery system.

Guidance in selecting appropriate doses of antibodies, cytokines, andsmall molecules are available (see, e.g., Wawrzynczak, 1996, AntibodyTherapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991,Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York,N.Y.; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy inAutoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al., 2003,New Engl. J. Med. 348:601-608; Milgrom, et al., 1999, New Engl. J. Med.341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792;Beniaminovitz, et al., 2000, New Engl. J. Med. 342:613-619; Ghosh, etal., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000, New Engl.J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

A preferred dose protocol is one involving the maximal dose or dosefrequency that avoids significant undesirable side effects. A totalweekly dose is generally at least 0.05 μg/kg body weight, more generallyat least 0.2 μg/kg, most generally at least 0.5 μg/kg, typically atleast 1 μg/kg, more typically at least 10 μg/kg, most typically at least100 μg/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0mg/kg, most preferably at least 2.0 mg/kg, optimally at least 10 mg/kg,more optimally at least 25 mg/kg, and most optimally at least 50 mg/kg(see, e.g., Yang, et al., 2003, New Engl. J. Med. 349:427-434; Herold,et al., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J.Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003, CancerImmunol. Immunother. 52:133-144). The desired dose of a small moleculetherapeutic, e.g., a peptide mimetic, natural product, or organicchemical, is about the same as for an antibody or polypeptide, on amoles/kg basis.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with diseaseand/or a reduction in the severity of such symptoms that will or areexpected to develop with said disease. The terms further includeameliorating existing symptoms, preventing additional symptoms, andameliorating or preventing the underlying causes of such symptoms. Thus,the terms denote that a beneficial result has been conferred on avertebrate subject with a disease.

The antibody of the present invention for therapeutic purposes isadministered in a therapeutically effective amount. As used herein, theterm “therapeutically effective amount” or “effective amount” refers toan amount of an anti-APRIL antibody or fragment thereof, that whenadministered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject is effective to prevent orameliorate the disease or condition to be treated. A therapeuticallyeffective dose further refers to that amount of the compound sufficientto result in amelioration of symptoms, e.g., treatment, healing,prevention or amelioration of the relevant medical condition, or anincrease in rate of treatment, healing, prevention or amelioration ofsuch conditions. When applied to an individual active ingredientadministered alone, a therapeutically effective dose refers to thatingredient alone. When applied to a combination, a therapeuticallyeffective dose refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously. An effective amount of therapeutic willdecrease the symptoms typically by at least 10%; usually by at least20%; preferably at least about 30%; more preferably at least 40%, andmost preferably by at least 50%.

Methods for co-administration or treatment with a second therapeuticagent are well known in the art, see, e.g., Hardman, et al. (eds.),2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics,10th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.), 2001,Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.),2001, Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.

The pharmaceutical composition of the invention may also contain otheragents, including but not limited to a cytotoxic, chemotherapeutic,cytostatic, anti-angiogenic or antimetabolite agents, a tumor targetedagent, an immune stimulating or immune modulating agent or an antibodyconjugated to a cytotoxic, cytostatic, or otherwise toxic agent. Thepharmaceutical composition can also be employed with other therapeuticmodalities such as surgery, chemotherapy and radiation.

The invention will now be further illustrated and supported withreference to the following non-limiting experiments.

Experiments

Experiment 1

Anti-APRIL Humanized Antibody Design

CDR Grafting

A unique antibody, hAPRIL.01A that binds human APRIL (WO2010/100056) waspreviously identified. The mouse hAPRIL.01A antibody was humanized byCDR-grafting technology (see e.g. U.S. Pat. No. 5,225,539 and Williams,D. G. et al., 2010, Antibody Engineering, volume 1, Chapter 21).

A strategy was designed in which first, human germline sequences wereidentified using IgBLAST (Ye J. et al., 2013, Nucleic Acids Res.41:W34-40). For the hAPRIL.01A VH, human germline sequence IGHV1-3*01(70.4% identity), and for the hAPRIL.01A VL, human germline sequenceIGKV1-16*01 (65.3% identity) was identified.

Next, a database was constructed containing all human maturatedsequences available in the IMGT database (release 201222-4: 161905entries, indexed 4 Jun. 2012) (Lefranc, M.-P. et al., 1999, Nucleic AcidRes. 27:209-212) identifying 90,401 individual sequences. Thesesequences were queried using TBLASTN (2.2.26+) to identify templatesequences that demonstrated the highest identify to hAPRIL.01A VH and VLsequences (SEQ IDs. 3 and 4, respectively). Three VH and seven VLsequences were selected that demonstrated a similarity score of 80% orhigher and that displayed similar CDR lengths, preferably identical tothose in hAPRIL.01A VH CDR1, CDR2, CDR3 (SEQ IDs. 5-7) and VL CDR1, CDR2and CDR3 (SEQ IDs. 8-10), respectively.

For the heavy chain, the frameworks encoded by GenBank (Benson, D. A. etal., 2013, Nucleic Acids Res. 41(D1):D36-42) accession # AF022000,AB363149, and AB063827 were selected for straight grafting of thehAPRIL.01A VH CDRs, resulting in the following cDNA constructs: SEQ IDs.11, 13, and 15, respectively. For the light chain, the frameworksencoded by GenBank accession # AX375917, DD272023, AB363267, AJ241396,DI152527, and DQ840975 were selected for straight grafting of thehAPRIL.01A VL CDRs, resulting in the following cDNA constructs: SEQ IDs.19, 21, 23, 25, 27 and 29.

An additional heavy chain sequence was designed based on the consensussequence from the alignment of the 25 best matching sequences (E-values5e-46 to 9e-43) from the TBLASTN result, resulting in the following cDNAconstruct: SEQ ID 17.

To determine the structural effects of humanization of frameworkresidues, a homology model of the hAPRIL.01A antibody was made usingWHATIF (Krieger E. et al., 2003, Methods Biochem Anal. 44:509-23). Thetemplates for the VH and VL chain, 2GKI (Kim Y. R. et al., 2006, J.Biol. Chem. 281: 15287-15295) and 2AEQ (Venkatramani L. et al. 2006, J.Mol. Biol. 356: 651-663) respectively were identified by a BLASTP search(Altschul, S. F. et al., 1990, J. Mol. Biol. 215:403-410) using theProtein Databank (www.rcsb.org, release June 2012; Berman H. M. et al.,2000, Nucleic Acids Res. 28:235-242). The VH and VL chains were combinedas Fab fragment using a MUSTANG alignment (Konagurthu A. S. et al.,2006, Proteins 64:559-574), which was guided by the 2AEQ template. Theconstructed homology model of hAPRIL.01A was used to select residuesthat are affected by humanization and could affect the functionality ofthe humanized construct and the evaluation was made whether or not toreplace selected residues: for the sixth VL template (VL15) it wasdecided to replace VL residues Y49 and Y87 by smaller S49 and F87.

Signal Peptide Identification

Using NCBI IgBlast (BLASTN) (Ye J. et al., 2013, Nucleic Acids Res.41(Web Server issue):W34-40) human germline repertoire matching themouse hAPRIL.01A VH and VL were identified and used to select thesecretion leader for the VH and VL: VH, based on germline IGHV1-3*01(NCBI accession # X62107), and VL, based on germline IGKV16*01 (NCBIaccession # X62109). The following VH secretion leader sequence“MDWTWRILFLVAAATGAHS” (SEQ ID NO: 58) coded by SEQ ID NO: 57 and the VLsecretion leader sequence “MDMRVLAQLLGLLLLCFPGARC” (SEQ ID NO: 60) codedby SEQ ID NO: 59 were used to express all humanized VH and VLconstructs.

An IgG4 version of humanized antibodies was produced, with thestabilizing Adair mutation (Angal S. et al., 1993, Mol Immunol. 30:105-108), where Serine 241 (Kabat numbering) is converted to Proline.

Experiment 2

Synthesis, Subcloning, Expression, Binding

Synthesis

cDNAs encoding humanized VH and VL constructs, SEQ IDs 11, 13, 15, 17,21, 23, 25, 27, 29, were codon-optimized using OptGene software (version2.0.6.0) and chemically synthesized by Baseclear. Next, sequences werecloned into the pUC57 vector (BaseClear), using a 5′-HindIII and 3′-ApaI(VH) or 3′-BsiWI (VL) restriction endonuclease cleavage site.

Subcloning

The humanized VH constructs were cloned into a pcDNA3.1(+) vector(Invitrogen) containing human IgG4 constant domains (CH1-CH3, GenBankaccession # K01316) that had been cloned into EcoRI and HindIIIrestriction endonuclease cleavage sites, using the above-mentionedrestriction endonuclease cleavage sites. The humanized VL constructswere cloned into a pcDNA3.1(+) vector (Invitrogen) containing a human CL(kappa) domain (GenBank accession # J00241) that had been cloned intoHindIII and EcoRI restriction endonuclease cleavage sites, using theabove-mentioned restriction endonuclease cleavage sites. Constructs weretransformed in Subcloning efficient DH5α competent cells (Invitrogen)according to the manufacturer's instructions. Plasmid DNA was isolatedusing the Qiagen Plasmid Midi Kit (QIAGEN) according to manufacturer'sprotocol. The integrity of the constructs was confirmed by DNAsequencing (Macrogen).

Expression and Binding

The plasmids encoding the VH and VL constructs were mixed in a 1:3 ratio(4 μg in total) and transiently expressed by transfection into HEK293Thuman embryonic kidney cells (HEK293T/17, ATCC-CRL-11268), usingLipofectamine 2000 transfection reagent (Invitrogen) following themanufacturer's instructions. Cell supernatants were harvested after 5days and tested for expression of antibody and binding to APRIL using anenzyme-linked immuno assay (ELISA). In these ELISAs, all incubationsteps were followed by a wash step with PBST (PBS with 0.01% Tween 20).Maxisorb 96-wells plates (Nunc) were coated with 0.5 μg/ml anti-FLAG(Sigma) or anti-IgG4 (Jackson laboratories) and incubated overnight at4° C. Subsequently the anti-FLAG coated 96-wells plates were incubatedwith FLAG-tagged human APRIL for 1 hour at room temperature. Next,supernatants and dilutions thereof were incubated for 1 hour, which wasfollowed by an incubation of 1 hour with mouse anti-human IgGHRP-conjugate (Southern Biotechnology).

Immunoreactivity was visualized with 100 μl TMB Stabilized Chromagen(Invitrogen). Reactions were stopped with 100 μl 0.5 M H₂SO₄ andabsorbances were measured at 450 and 620 nm.

Experiment 3

Purification and Stability

Purification

A subset of humanized antibodies described above was selected forfurther analyses. Again, plasmids encoding the VH and VL constructs weremixed in a 1:3 ratio (32 μg) and transiently expressed by transfectioninto (8*10⁶) HEK293T human embryonic kidney cells (HEK293T), usingLipofectamine 2000 transfection reagent (Invitrogen) according to themanufacturer's instructions. Supernatants were harvested (10 ml) andantibodies were purified using MabSelect Sure Protein A resin accordingto the manufacturer's instructions (GE Healthcare). Buffer was exchangedfor PBS using Zeba desalting columns (Thermo Scientific). Theconcentration of purified antibodies was determined based on OD280(Nanodrop ND-1000). The binding of the purified antibodies to APRIL wasestablished using the above described APRIL ELISA. The blockingcapability of the humanized antibodies with BCMA and TACI receptors, wastested in a competition ELISA. In these ELISAs, all incubation stepswere followed by a wash step with PBST (PBS with 0.01% Tween 20).Maxisorb 96-wells plates (Nunc) were coated with 0.5 μg/ml Fc-BCMA (R&DSystems) or Fc-TACI (R&D Systems) and incubated overnight at 4° C. Next,humanized antibodies and dilutions thereof were incubated, premixed withFLAG-tagged APRIL, for 1 hour, which was followed by an incubation of 1hour with anti-FLAG HRP-conjugate (Sigma). Immunoreactivity wasvisualized with 100 μl TMB Stabilized Chromagen (Invitrogen). Reactionswere stopped with 100 μl 0.5 M H₂SO₄ and absorbances were measured at450 and 620 nm. Calculated EC₅₀ and IC₅₀ representing the concentrationat which 50% of the total binding signal or blocking is observed arerepresented in Table 5.

TABLE 5 EC50 values, binding to APRIL. IC50, blockade of APRIL bindingto BCMA-Fc. C4-hAPRIL.01A was used as experimental reference in eachELISA. VH.VL combination EC50 (nM) IC50 (nM) C4-hAPRIL.01A 5.7 7.6VH11.VL15 120.8 4.3 VH12.VL15 19.8 n.c. VH13.VL15 223.4 n.c. VH14.VL1548.3 n.c. n.c. indicates inhibition, but no IC50 could be calculated dueto improper fitting.

For blockade of APRIL binding to TACI-Fc similar blocking effects wereobserved.

Surprisingly, combinations of VH11-VH14 with VL10-VL14 did not shownano- or micromolar EC50 values and only the combination of the selectedVH framework sequences with framework sequences of VL15 resulted inantibodies having functional APRIL binding properties.

Stability

To determine the effect of humanization on the stability of theantibodies, humanized antibodies were exposed to a range of temperaturesfor 10 minutes. Purified antibodies were diluted to 3.16 μg/ml anddilutions thereof in PBS. Next, these solutions were exposed to 65° C.or 70° C. and residual binding after heat treatment of the antibodieswas measured using the FLAG-tagged APRIL ELISA assay as described before(see Table 6).

TABLE 6 Residual binding of (humanized) antibodies to FLAG-APRIL asdetermined by ELISA. Binding is measured at three concentration: 3.16, 1and 0.316 μg/ml. % Binding at 65 or 70° C. is expressed as % bindingobserved for each of the antibodies at Room Temperature (=100%).concentration 65° C. c4- hAPRIL.01A hAPRIL.01A 11.15 12.15 13.15 14.153.16 27.1 42.8 63.1 66.4 76.2 58.6 1 8.4 10.4 55.1 49.8 44.4 45.2 0.31613.7 18.8 74.6 74.3 66.1 63.7 Concentration 70° C. c4- hAPRIL.01AhAPRIL.01A 11.15 12.15 13.15 14.15 3.16 6.7 6.6 59.1 50.2 80.5 50.0 14.9 7.4 62.4 51.8 47.4 42.5 0.316 11.2 16.9 82.0 70.3 65.0 63.1

Experiment 4

Improvement of Binding, Blocking and Stability by Back Mutations andVernier Residues

Improvement of Binding and Blockade by Back Mutations

Analyses on sequence and structural level were performed to understandthe molecular basis for the differences in binding and blockade of thedifferent VH/VL combinations. A homology model of the humanized antibodywas made, as described before. The template selected for both VH and VLwas 3HC4 (Jordan J. L. et al., 2009, Proteins 77: 832-841). On the basisof careful analysis of the created model, the inventors of the presentinvention postulated that residue S72 in the selected VH chains isimportant for the orientation of the CDR2 loop. In order to investigatethis postulation, mutation R72S was introduced in VH 14, which resultedin VH 14_1, SEQ ID 32 coded by the nucleotide sequence of SEQ ID 31.Antibody 14_1.15 was tested for binding and blockade as describedbefore. As represented in Table 7, binding and blockade of antibody14_1.15 are improved relative to the binding of antibody 14.15 as shownin table 5.

TABLE 7 Binding to APRIL and blockade of APRIL binding to BCMA-Fc ofantibody 14_1.15. hAPRIL.01A and C4-hAPRIL.01A were used as experimentalreference in each ELISA. VH.VL combination EC50 (nM) IC50 (nM)VH14_1.VL15 1.29 ± 0.16 2.63 ± 0.55 C4-hAPRIL.01A 0.35 ± 0.13 0.77 ±0.22 hAPRIL.01A 0.16 ± 0.14 0.46 ± 0.35

For blockade of APRIL binding to TACI-Fc similar IC50 values wereobtained.

Vernier Residues

Analyses on sequence and structural level were performed to furtherimprove binding and blockade of antibody 14_1.15. A homology model ofthis hAPRIL.01A analogue was made, as described before. The selectedtemplate for the VH chain was 2GKI and for the VL chain 4GMT (Lee P. S.et al., 2012, PNAS 109: 17040-17045), combined as a Fab fragment guidedby template 2AEQ.

Residues close to the CDRs were studied in detail, since they couldaffect the loop conformation. In the analysis, the inventors identifieda number of potentially relevant Vernier residues (Foote J. et al.,1992, J. Mol. Biol. 224:487-499). In order to evaluate their relevancythey were substituted with the mouse amino acid.

Introduction of mutation M70I resulted in VH14_1C (SEQ ID 33, 34)mutation T74K is present in VH14_1D (SEQ ID 35, 36), and mutation Q1Eresulted in VH14_1E (SEQ ID 37, 38). The combined mutation of R67K andV68A resulted in VH 14_1G, SEQ ID 39, 40. The antibodies were tested forbinding, blockade, and stability as described before. As represented inTable 8, surprisingly binding and blockade are improved with a factor 2to 3. In particular the mutations introduced in antibody VH14_1G.VL15surprisingly present a considerable improvement.

TABLE 8 Binding and blockade of antibody 14_1.15 and vernier zonemutants. hAPRIL.01A was used as experimental reference in each ELISA.VH.VL combination EC50 (nM) IC50 (nM) VH14_1.VL15 1.29 ± 0.16 2.63 ±0.55 VH14_1C.VL15 7.04 ± 2.23 5.26 ± 0.08 VH14_1D.VL15 1.95 ± 0.28 0.96± 0.38 VH14_1E.VL15 2.67 ± 0.28 1.74 ± 0.32 VH14_1G.VL15 0.78 ± 0.161.35 ± 0.39 hAPRIL.01A 0.16 ± 0.14 0.46 ± 0.35

In addition, the stability of the substituted humanized antibodies wasimproved as determined using thermostability studies as described inExample 3 (see Table 9).

TABLE 9 Residual binding of (humanized) antibodies to FLAG-APRIL asdetermined by ELISA. Binding is measured at three concentration: 3.16, 1and 0.316 μg/ml. % Binding at 65 or 70° C. is expressed as % bindingobserved for each of the antibodies at Room Temperature (=100%).concentration 65° C. 14.15 14_1.15 14_1C.15 14_1D.15 14_1E.15 14_1G.153.16 59.0 59.9 86.3 75.3 82.3 94.5 1 47.5 46.1 44.7 39.8 39.7 31.3 0.31667.5 71.0 59.1 49.4 52.0 34.2 Concentration 70° C. 14.15 14_1.1514_1C.15 14_1D.15 14_1E.15 14_1G.15 3.16 58.6 38.0 99.4 84.3 78.8 78.2 155.1 38.3 43.8 32.7 28.0 16.9 0.316 79.1 63.0 59.1 45.6 43.3 23.8

Experiment 5

14_1G.15 Demonstrates More Efficacious In Vivo Inhibition.

To demonstrate an in-vivo blocking effect of the APRIL analogueantibodies on APRIL function, we examined the ability of the antibodiesto block the NP-Ficoll induced humoral response in mice. The mice usedwere 8-10 week old APRIL transgenic (TG) mice and wildtype (WT)littermates, both on a C57BL/6 background. The APRIL transgenic miceexpress human APRIL under the Lck-distal promoter, which directstransgene expression to mature thymocytes and peripheral T lymphocytes(Stein et al., 2002, J Clin Invest 109, 1587-98). The mice were bred inthe animal facility of the Academic Medical Center and the experimentwas approved by the institutional ethical committee. The mice weredivided into several groups and treated as follows: WT mice were treatedwith PBS (200 μl) and 3 groups of APRIL transgenic mice were treatedwith the following molecules: hAPRIL.01A or 14_1G.15 (200 μg/mouse onday −1 and day 3 in 200 μl PBS) or PBS. On day 0, mice were immunizedwith NP-Ficoll (day 0; 100 μl i.p. with 250 μg of the immunogen). Bloodwas collected via tail vein at day −1, 3, 7, 10.Anti-(4-hydroxy-nitrophenacetyl) (NP)-specific antibodies (IgM, IgG andIgAa/2) were assayed by ELISA using diluted sera as previously described(Hardenberg et al., Immunol Cell Biol, 86(6):530-4, (2008); Guadagnoliet al., 2011, Blood 117(25):6856-65). Briefly 96-well ELISA plates(Greiner) were coated with NP-BSA at 5 μg/ml (Biosearch Technologies) insodium carbonate buffer (pH 9.6) overnight at 4° C. The wells wereblocked with 1% BSA for 1 hr at 37° C. and incubated with diluted serafor 2 hrs at room temperature. HRP-conjugated isotype specificantibodies (Goat anti-mouse IgG, IgA and IgM—from Southern Biotech) wereused as revealing antibodies. All dilutions were made in PBS/BSA1%/Tween 20 0.05%. As apparent from FIG. 1, both hAPRIL.01A and 14_1G.15inhibited the T-cell independent B-cell responses in vivo. hAPRIL.01Ainhibited this response less efficacious then 14_1G.15. PBS and mouseIgG1 as an isotype-matched control, did not affect the IgA, IgM and IgGanti-NP response.

1-7: (canceled)
 8. A humanized antibody having (i) light chaincomplementary determining regions of SEQ ID NO: 8, SEQ ID NO: 9, and SEQID NO: 10, (ii), heavy chain complementary determining regions of SEQ IDNO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and framework regions having atleast 90% sequence similarity to the framework regions of SEQ ID NO: 50and SEQ ID NO: 52, wherein the antibody binds to human “Aproliferation-inducing ligand” (“APRIL”) protein.
 9. A humanizedantibody having a light chain of SEQ ID NO: 50 and a heavy chain of SEQID NO:
 52. 10. A method according to claim 8, wherein at least 90%sequence similarity is at least 90% sequence identity.
 11. A methodaccording to claim 8, wherein at least 90% sequence similarity is atleast 95% sequence similarity.
 12. A method according to claim 11,wherein at least 95% sequence similarity is at least 95% sequenceidentity.